Human Interferon-γ (IFN-γ) ELISPOT Kit

Materials Provided

Product Overview

Interferon-γ (IFN-γ) is the only type II interferon. This proinflammatory cytokine is secreted by activated T cells and NK cells. It activates macrophages and endothelial cells and regulates immune responses by affecting APCs, T cells, and B cells. Production of IFN-γ by helper T cells and cytotoxic T cells is a hallmark of the Th1-type phenotype. Thus, high-level production of IFN-γ is typically associated with effective host defense against intracellular pathogens. The Human IFN-γ ELISpot assay is designed for the detection of human IFN-γ secreting cells at the single cell level and can be used to quantitate the frequency of human IFN-γ secreting cells. ELISpot assays are highly reproducible and sensitive, and can be used to measure responses with frequencies well below 1 in 100,000. ELISpot assays do not require prior in vitro expansion of T cells and they are suitable for high-throughput analysis using only small volumes of primary cells. As such, ELISpot assays are useful tools for research in vaccine development, cytokine secretion and the monitoring of various clinical trials.

Application

This kit is used to detect IFN-γ at the cellular level.

It is for research use only.

Reconstitution

Please see Certificate of Analysis for details of reconstitution instruction and specific concentration.

Storage

The unopened kit is stable for 12 months from the date of manufacture if stored at 2°C to 8°C.

Assay Principles

ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. The Kit consists of Pre-coated Anti-IFN-γ Antibody Microplate, Positive Stimulus, Biotin-Anti-IFN-γ Antibody, Streptavidin-HRP, AEC and buffers.

Your experiment will include 6 simple steps:

a) A monoclonal antibody specific for human IFN-γ has been pre-coated onto a polyvinylidene difluoride (PVDF)-backed microplate.

b) Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37 °C CO2 incubator for a specified period of time.During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted IFN-γ.

c) After washing away any cells and unbound substances, a biotinylated antibody specific for human IFN-γ is added to the wells. Following a wash to remove any unbound biotinylated antibody, HRP conjugated to streptavidin is added.

d) Wash the plate and add the Streptavidin-HRP diluted by Dilution Buffer to the plate.

e) Wash the plate and add AEC. A reddish-brown colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual IFN-γ secreting cell.

f) SThe spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.

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